Oxidation of steroids by mycobacteria



2,905,592 OXIDATION OF STEROIDS BY MYCOBACTERIA Gilbert M. Shull,Huntington Station, and Donald A. Kita, Jackson Heights, N.Y., assignorsto Chas. Pfizer & Co., Inc., New York, N.Y., a corporation of DelawareNo Drawing. Application July 27, 1955 Serial No. 524,817

12 Claims. (Cl. 19551) This invention is concerned with the oxidation ofcertain steroid compounds by microbiological means. In particular, it isconcerned with the oxidation of certain 3-keto-4-unsaturated steroidcompounds by means of certain microorganisms or oxidizing enzymesproduced by these microorganisms.

This application is a continuation in part of applications Serial Number431,619, filed May 21, 1954; Serial Number 483,842, filed January 24,1955; and Serial Nlnnber 484,828, filed January 28, 1955, by Gilbert M.Shull et 21. It is also a continuation-in-part of application Serial No.276,678, filed March 14, 1952, and now abandoned.

As is disclosed in the above mentioned applications, by subjecting a3-keto-4-unsaturated steroid compound to the action of a species of thegenus Mycobacterium, there is produced a compound which is a3-keto-l,4-unsatu rated steroid. Some nuclear oxygenation also takesplace, chiefiy in the 14 position when this position is available. Themain product of the oxidation, however, is the dehydrogenation product.It is thus possible by the use of organisms of the genus Mycobacteriumto produce compounds having a 3-keto group and unsaturated at the 1 and4 positions. Certain compounds of this type are extremely valuablebecause of their biological activity, and it is therefore of great valueto have a simple, one step method of dehydrogenation at the 1,2positions.

This invention is the first case in which dehydrogenation in the A ringof a steroid has been produced by the use of microorganisms without thesimultaneous oxidative cleavage of a side chain present in the 17position. This invention is also the first instance in which an organismof the genus Mycobacterium has been used in a commercial chemicalreaction and is indeed believed to be the first time that any use hasbeen found for organisms of the family Mycobacteriaceae.

It has now been found that on contacting a 3-ket0-4-unsaturated steroidcompound with the oxidizing activity of a species of the genusMycobacterium there is produced oxidation of the steroid compound. Thechief chemical reaction is one of dehydrogenation in the 1,2 positions,thereby producing a double bond between these positions. Someoxygenation also occurs at the same time, chiefly in the 14 positionwhen this position is available. The term nuclear oxidation includesboth dehydrogenation and oxygenation. It is possible that sometimes bothtypes of oxidation, i.e. dehydrogenation and oxygenation, occur in thesame molecule, but this is not generally the case to a large extent. Theprocess of this invention, i.e. the contacting of a suitable steroidcompound with the oxidizing activity of a species of the genusMycobacterium, may be carried out by bringing the chosen steroidcompound in aqueous solution or suspension into contact with either anactively growing culture of Mycobacterium, with cells of Mycobacteriumremoved from the growing culture and suspended in a suitable medium, orby contacting the steroid with extracts of oxidizing enzymes obtainedfrom the culture of a species of the genus Mycobacterium.

, oxidation of the steroid nucelus.

2,905,592 Patented Sept. 22, 1959 An object of this invention is aprocess for the preparation of oxidized steroids, particularly 3-keto-Asteroids, from the corresponding 3-keto-4-unsaturated compounds. Afurther object of this invention is an economical process for thisdehydrogenation reaction which may be readily scaled up for large volumeproduction. Further objects of this invention will be apparent from thefollowing discussion.

A variety of 3-keto-4-unsaturated steroid compounds may be usedas'starting materials for the reactions of this invention. These includesuch materials as:

Compound F (hydrocorfisone) 9a-fluoro-Compound F Compound E (cortisone)l4a-hydroxy-Compound F 17a-hydroxyprogesterone 16-dehydroprogesteronell-ketoprogesterone A9(ll)dehydro-Compou11d S In general this process ismost applicable to 3-keto-A steroid compounds having from 18 to 21 atomsin the carbon skeleton. The products of the reaction may be detected bycareful comparison of paper chromatograms of the products formed by thereaction of this invention with known steroid compounds. This method hasbeen tested on a variety of compounds and is known to give reliableresults. Reports of this method are available in the chemicalliterature.

In carrying out this invention various species of the genusMycobacterium may be used for bringing about These are available frompublic culture collections, for example the American Type CultureCollection of Washington, DC. These include known species such asMycobacterium species 607, M. berolinense, M. lacticola, M. thamnopheos,and the species M. smegmatis ATCC 101. Particularly useful are organismsof the species M. phlei, a living culture of which has been depositedwith the American Type Culture Collection of Washington, DC, Where ithas been given the number ATCC 354. The various species of Mycobacteriumvary considerably in the speed and ease with which dehydrogenation isproduced by the cells of the organisms or by products formed duringtheir growth. We have found that certain strains of M. 'phlei and M.smegmatis are particularly active in bringing about the reaction of thisinvention. The various mycobacteria vary somewhat in their requirementsfor nutritional media. By a minimum of single testing, however, it ispossible to determine the composition of suitable media, as well as theoptimum conditions for operation, such as pH, rate of aeration, rate ofstirring and so forth. In a like manner, the optimum conditions for thedehydrogenation of a particular steroid may be found.

There are several procedures which may be used in the dehydrogenation ofsteroid compounds according to this invention. In the first of these,nutrient media are seeded from slants of the selected Mycobacterium.Such a medium may consist, for instance, of a mixture of a standardbacteriological nutrient broth base, together with glycerol. It has beenfound that a neutral, surface-active agent, such as a polyoxyethylenederivative of a sugar alcohol-fatty acid ester (eg. Tween when added ina minor proportion to the medium, is helpful. The addition of the aminoacid asparagine is also helpful. The cultivation of mycobacteria hasbeen described in considerable detail in many publications. The seeded,sterile, nutrient solutions may be grown in shake flasks for two tothree days to provide inoculum for larger vessels, and in turn, thelarger, stirred, aerated vessels may be used for the inoculation of fullproduction-scale vessels for submerged fermentation. The same medium-ofthe type described above may be used for the large-scale oxidation ofsteroidsaccording-to this invention. Considerable variation may, ofcourse, be made in the medium. In general there is required acarbohydrate, a source of organic nitrogen, mineral salts and varioustrace metals.

As pointedout above, rather than'conducting the oxidation of theselected steroid compound in the presence of the whole fermentationproduct, cells may be removed fromgrowing cultures and these may beresuspended in a medium -which has been designated the enzyme reactionmixture. Such a reaction mixture may consist, for .instance, of asolution which is 0.01 molar in sodium fumarate or other hydrogenacceptor and in magnesium sulfate and.0.03 molar in sodium citrate. Ithas been found that the presence of-a certainamount of adenosinetriphosphate, e.g.:0.125%, is alsoquite useful. Centrifuged, washedcells of the chosen Mycobacterium may be suspended in this type ofreaction mixture, which is adjusted to a pH of about 6, for example withcitric acid. After addition of the steroid compound which it is desiredto oxidize, the mixture may be incubated at about 37 C., and samples maybe removed from time to time to determine the point at which maximumconversion of the steroid has taken place. In general, this occurs afterabout one to several days. We have found that the cells from about 100milliliters of the stirred, aerated mycobacteria cultures may besuspended in about 20 milliliters ofan enzyme reaction mixture forsuitable results. Considerable variation may be made in theseproportions.- The steroidcompound may be used in a proportion of about25 to about 200 milligrams/100 milliliters of the enzyme reactionmixture. The compound in solid form is merely added to the medium afteradjustment of the pH. The flasks are stoppered with cotton so that theyare exposed to the air during the incubation. We prefer to-use a smallvolume compared to the volume of flask, for instance, 20 milliliters ina l25-milliliter Erlenmeyer flask. Alternatively, the mixture may bestirred and aerated. Ingeneral, at least a hydrogen acceptor, a divalentmetal particularly magnesium, and a buffer are required in-the medium.

Rather than removing the Mycobacterium cells and carrying out thereaction of this invention in an enzyme reaction mixture, the steroidcompoundmay be added directly to a sterilized portion of nutrientmedium, such as is described above, and the medium is then seeded withthe chosen mycobacteria. Approximately the same proportion of chosensteroid compound may be used in this case also. Samples of the agitated,aerated mixture may be removed at intervals for determination of theconversion of the steroid compound to the oxidized products. The mixtureis maintained at between 2537 C. or higher during the growth of thecells and the conversion of the steroid. In general, about twoto sevendays is required for maximum production of the oxidized compounds.Alternatively, the growth of the cells may be established beforeaddition of the steroid.

A third method which is also very useful for the oxidation of theselected steroid compounds involves the use of oxidizing enzymesproduced by the mycobacteria. These may be prepared by a variety ofmethods from the cells of the chosen organisms. These materials may bereleased from the cells by several different procedures. These includegrinding, particularly with abrasive materials such as powdered glass orsand, which serves to break the cell walls and release the essentialmaterials. A second method is by autolysis. The cells may be removedfrom the medium in which they are grown. They are then washed andsuspended in water. The water may be covered with a thin layer oftoluene to prevent contamination, and the mixture is allowed to stand ata temperature of from about 20 to about 50 C. The cells disintegratewithin one to several days and the cell residue may be removed byfiltration, for instance through a Seitz filter or through a sinteredglass bacterial filter. A third'method for preparing cell-freeelaborationprod- '4 ucts of the mycobacteria useful for the reactions ofthis invention is by repeated, rapid freezing and thawing of thecellular material. Another method is by the use of ultrasonic energy torupture the cells. One further meth- 0d of use for the same purpose isby the use of a watermiscible solvent and, in particular, acetone. Thecells, when placed in such a solvent, are ruptured and an extract ofthefdesired enzymes is obtained. The mycobacteria enzymes may be usedfor theoxidation of 3-keto-4- unsaturated steroid compounds in mediasimilar to those used with the grown cells that is, one containingahydrogen acceptor such as fumarate, a buffer and, in some cases, abivalent metal, particularly magnesium, as well as a minor proportion ofadenosine ,triphosphate. The

cell-free oxidizing enzymes of mycobacteria may be used in mediaindicated above at a temperature of about 20 to about 40 C. In general,the oxidation of the desired steroid compounds is brought about in aperiod of from a few hours to several days. The optimum time andtemperatureand other conditions may readily be determined by a minimumof experimentation. Detailed descriptions of suitabie mediafor both theuse of isolated, resuspended cells and of cell-free elaboration productsare givenin the textbooks Manometric Technique in Tissue Metabolism, byW. W. Umbreit etalQ, Burgess Publishing Company, Minneapolis (1949), andRespiratory Enzymes, by H. Lardy, Burgess Publishing Company,Minneapolis 1949) The most convenient method for following the course ofthe oxidation reaction is by means of paper chromatography. Othermethods may also be used for this purpose as noted above. In the paperchromatography method, aluminaimpregnated-filter paper is prepared bydippinglarge sheets of thepaper (Whatman No. S t-27" x 22 /2"-sheets-filter paper is particularly useful) in an aluminum sulfatesolution (13 grams/ milliliters). The paper is drained, exposed toanatmosphere of am monia for 15 hours and the sheets are then washedcontinuously for six hours with tap water. The sheets are then drained,ironed at a moderate temperature to obtain a flat surface, and arestored at room temperature for 24 hours prior touse. Various solventsystems are used in the development vof the paper chromatograms. Thesehave-been designated as follows:

SystemI-zHexane System IIHexane-ether (19 volumes to -1 volume) SystemIII=Hexane-ether (16 volumes to 1 volume) System IV-Ether Fivemicroliter quantities of solutions containing the steroids are placed onthe paper along aline three centimeters from one edge of the paper andthe spots are spaced two centimeters apart. The edge of each paper isstapled to the opposite edge to form cylinders and each paper is placedin a glass jar of appropriate size. They are exposed to the vapors ofthe solvent system for one hour at 4-8 C. The jars are then covered withtransparent plastic lids, which are sealed with high vacuum siliconegrease after sufficient solvent has been placed in the bottom of thejar. The solvent is carried up the paper by capillarity and, in passingthe spots where the steroids have been deposited the various componentsof these products are moved up .the paper ata greater or lesser ratedepending upon the structure of the individual compound. The developmentof the sheets is carried out in acold room atabout 48 C. until thesolvent front reaches .a point about eight centimeters from the top ofthe paper :sheet. This generally takes about .four to five hours. Thepaper cylinders are then removed and dried at room-temperature. V

One further system has been used for-the comparison ofthe varioussteroid products. Thisis-known as System V. 'It consists of benzenesaturated with'water. With this system, however, untreated {Whatman No.54 filter paper sheets aroused rather than the alumina impregnatedsheets, and cylinders of the. papers are prepared of 8" x 18" sheets.Before chromatographing the products, the atmosphere of the jars issaturated with water by means of steam. The benzene saturated with wateris poured in the bottom of the jar and the lid is sealed. Development isallowed to occur at room temperature until the solvent front is close tothe top of the paper (three to four hours). The paper is then removedand dried at room temperature. The paper chromatography methods ofZaffaroni and of Bush, reported in the literature, are also adaptablefor use here.

Various methods may be used to detect the various steroids on the paperchromatograms. The first of these methods entails no chemical reactionwith the separated steroids and may be used before the use of either ofthe other methods. In this method, the sheets are examined With afluorescent scanner as described by Haines et al., FederationProceedings, volume 9, p. 180 (1950). The steroids having an0a,;3-l11183t1118i6d ketone structure and certain dienic steroids appearas dark spots when viewed through the phosphorescent screen. These maybe outlined on the sheets in pencil to establish their position.Standard, known samples of the steroids may be run side by side withmaterial prepared according to this invention. In this way it ispossible to distinguish newly produced oxidized steroids. A secondmethod for locating the steroid compounds on the paper sheets is bytreatment of the sheets with a atmosphere of chlorine for 20 minutesandthen spraying the sheets with a solution of 380 grams of antimonytrichloride in 100 milliliters of acetic anhydride. The solution isprepared fresh daily. The sprayed sheets are heated at 90100 C. untildry and immediately examined in a dark room with a sun lamp usingaCorning No. 9863 filter. Spots formed on the paper may be outlined withpencil and the relative position with respect to known samples of thevarious steroids may then be established.

An alternative method for locating certain of the steroid products is bymeans of the reagent described by Burton et al., J. Biol. Chem., volume188, p. 763 (1951), which involves the spraying of the paper with analcoholic potassium hydroxide solution of triphenyltetrazolium chloride.Steroids having a 2l-hydroxy-20-keto side chain appear as red spots ondrying the paper. The spots are immediately outlined in pencil since thecontrast with the background soon disappears. By running paperchromatograms with a group of known oxidized steroid compounds, it hasbeen possible to distinguish the products formed by the process of thepresent invention and prove the oxidation which takes place during thereaction. The various solvent systems have been used to establish, withcertainty, the nature of the materials which have been formed. Inconducting the paper chromatographic work, a value known as Rf has beenused to identify the position of the various steroid compounds on thepaper chromatograms. The R value for any given compound using a specificsolvent system is the ratio of the distance travelled by the compoundfrom the spot at which it was applied on the paper sheet as compared tothe distance travelled by the solvent front under exactly the sameconditions. The R value will, of course, vary with different types ofcompounds, with the different solvent systems, with variations intemperature, and with variations in the nature of the paper used.However, the same compound under the same set of conditions Will givethe same Rf and thus serve to indicate the identity of the particularproduct. A standard table of R, values may be prepared for use with agiven solvent system under a specific set of conditions. These valueshave been listed in a publication of Gilbert M. Shull et a1. (Archivesof Biochemistry and Biophysics), vol. 37, p. 186 (1952).

The products of the new method described in this application may beisolated from aqueous solution by extraction with variouswater-immiscible organic solvents. Lower halogenated hydrocarbons,such'as chloroform,

are particularly useful. After extraction, the solvent may be removed bydistillation and the solid product is then isolated. This material maybe further purified by recrystallizattion procedures from organicsolvents or by chromatography, for instance on alumina columns or onother suitable solid absorbent materials. The use of a silicagel-ethanol column with a 98% to 2% by volume mixture of, methylenechloride and ethanol as a developer has been found particularlyadvantageous. Methods for the separation of products of this nature havebeen reported previously in the literature. For some uses the productsneed not be separated, but the crude mixture may be used as such. It hasbeen found advantageous in some cases to acylate the crude products andwork with the resulting esters which are somewhat more stable.

A variety of 3-keto-4-unsaturated steroids are possible startingmaterials for the reactions of this invention. These include suchwell-known compounds as testosterone, progesterone, and ReichsteinsCompound S. The oxidized products are useful as intermediates in thesynthesis of other useful compounds. For example, the dehydrogenatedproducts which contain unsaturation at the 1,2 position, along with the3-keto group and the 4,5- unsaturation originally present in thestarting material, are especially susceptible to Inhoffen aromatization.This gives rise to a group of derivatives of estrone. In the case of thedehydrogenation product of Compound S, side chain cleavage to give a17-keto group way readily be accomplished by standard means, e.g.oxidation With chromic acid, and when the product of that reaction isaromatized by the Inhoffen reaction, e.g. heated to a high temperaturein a hydrocarbon solvent, the very valuable compound estrone isproduced. The introduction of oxygen functions into the nuclei of thesteroids Which have been oxygenated has imparted to these molecules newcenters of chemical activity, and has greatly enhanced their versatilityin undergoing further chemical reactions. Very desirable changes insolubility are also effected by the introduction of hydroxyl groups.

In addition to the utility mentioned above, many of the compoundsproduced by this reaction are extremely valuable because of their greatbiological activity. For example, when hydrocortisone is treated Withmycobacteria according to the process of this present invention, thecompound formed is prednisolone, which is of great utility and hasadvantages over hydrocortisone in the treatment of rheumatoid arthritis.When cortisone is treated with mycobacteria, there is produced thecompound known as prednisone which also has great utility in thetreatment of rheumatoid arthritis. It has also been found that other3-keto-A steroids possess great activity as adrenocortical hormones andare useful for the same type of therapy as hydrocortisone. Manynaturally occurring steroids, and steroids readily prepared fromnaturally occurring ones, have a 3-keto-A structure, but no 3-keto-Acompounds are readily available as raw materials. For this reason, theprocess of this invention whereby it is possible to transform a 3-keto-Acompound.

having from 18 to 21 atoms in the carbon skeleton into a 3-keto-Acompound in one single step in large scale commercial production is oftremendous value.

The following examples are given by way of illustration and are not tobe considered as limitations of this invention, since as many apparentlyWidely different embodiments 0f the present invention may be madewithout departing from the spirit and scope hereof.

A living culture of the organism employed in Examples I through IVhereinafter, namely Mycobacterium smegmazis (Pfizer Culture CollectionNo. 2), has been deposited with the American Type Culture Collection,2029 M Street, NW., Washington, DC, and added to its permanentcollection of microorganisms as ATCC 12,549..

7 EXAMPLE I A culture of Mycobacteriumsmegm'atis' (Pfizer- Cultu'reCollection No: 2)- grown on solid agar medium, was

added to 50 milliliters of nutrient solution having the The medium wassterilizedbefore' seeding. This mixture was placed in a" 250-milliliterErlenmeyer flask and the flaskwas shaken at about 27 C. for three days.The contents-of one shake flask were used to inoculate tlwo liters ofthe same sterilized medium inc. 4 liter vessel equipped for conductingsubmerged fermentation. The medium was seeded and the mixture wasstirred and aerated-under sterile conditions.- The stirrer was operatedat aspeed of 1750 r.p.m. and aeration was continued throughout 48 hoursat' a rate of one volume of sterile air per'volume of medium per minute.The cells that had formed in the medium were centrifuged, and washedwith water.

An enzyme reaction mixture wasprepared containing 0.01 molar sodiumfumarate, 0.01 molar magnesium sulfate, 0.03' molar sodium citrate and0.125% adenosine triphosphate; Twenty milliliters of this medium wasplaced in a 125-milliliter Erlenmeyer flask. After the pH had beenadjusted to 6.0 with citric acid, to the reaction-mixture was added 25milligrams of Compound S acetate. The reaction mixture was seeded withthe cells'from 100 milliliters of the submerged fermentation' and themixture was incubated at 37 C. Samples were removed at intervals, andthe steroids present were extracted into chloroform. After separationof: the organic phase, the solvent was removed and the product wasdissolved'in ethanol; Samples ofthe ethanol solution wereplace'd onpaper sheets using the chromatographic technique described above. of sixhours some oxidation of the nucleus of the Com pound S acetate hadoccurred. The reaction proceeded quite-rapidly and in 24 hoursappreciable conversion had occurred; At the end of 48' hours, a maximumoxidation of the steroid nucleus had taken place. Paper chromatogramsrun using solvent SystemV gave the following results'. The figures givenin the table are R values and the names in parentheses are the steroidcompounds to which each of the spots on the paper chromatogram conforms.These tmts were made 48 hours after inoculation of the medium withcells.

R ValueS on System V:

(Compound S acetate) 1.0 (Compound S) 0.58 (A -dehydro-Compound S andl4a-hydroxy- Compound'S) 0.30 0.20

(Not yet identified) EXAMPLE H To a four liter Pyrex glass vesselequipped for conducting submerged aerated fermentation was added twoliters of the following medium (known as Turfitts medium):

Ferrous sulfate heptahydrate 0.00001 Calcium carbonate 0.5

The aqueous medium was sterilized and with 0.25 gram of Compound Sacetate. The mixture was then: seeded with 100 mls. of a culture ofMycobacte'rium smegmatis (Pfizer Culture Collection No. 2)

grown in a shake flask on-nutrient broth. The mixture- It was" foundthat atthe end then treated by means of paper chromatograms usingthree=dififerent systems. The following table indicates the R; valuesofthevarious systems. The compoundsv to which these spots correspond aredesignated at theside of the' tables Rt Values System Steroid Compounds0 III IV Compound S Compounds acetate." Compound-S; A -dehydro-ClompoundS and 14m-hydroxy-Compound S Another system of paper chromatography gavebetter results, especially in the separation of the A -dehydro- CompoundS and l4 t-hydroxy-Compound S, each of which had the R; value of 0.45when System V was employed. This other systemwasemployed inadescending'chro'matograph' instead'of an ascending one as thosepreviously described. The paper was impregnated with a mixture of-volumes of methanol and 20 volumes of water. The atmosphere in the jarwas saturated with' the vapor of toluene which had been saturated withwater. Developing was accomplished by means of a mixture of l00'volun1esof toluene and 20 volumes of ethyl alcohol;

By use of this system, the following results were obtamed.

R values:

(Compound S) 1.00

These R values were obtained by assigning a value of 1.00 to thedistance traveled by Compound S, and to' each of the other compounds avalue equal to the ratio of the distance traveled by-that' compound tothe distance traveled by Compound S; Both the solvent front and Compound8* acetate went over the entire length of the sheet and off the paper.

The mixture was finally separated by successive applications to silicagel-ethanol columns. When fermentation was completed, the fermentationmixture was extracted with" chloroform, and evaporated to a volume ofabout 20 ml. This concentrated extract was then applied to a silicagel-ethanol column, and eluted with methylene chloride-alcohol mixtureuntil most of the n -dehydro-Compound S had been removed. The column wasthen eluted with methanol" to remove all the'remaining' steroids. Thisprocedure was repeated several times, each time starting with theextract of a fermentation conducted as described above. These methanoleluates' were thencombined, evaporated to dryness and dissolved in20'ml; of chloroform. This chloroform solution was then applied to a silicagel-ethanol column and eluted with the methylene chloride-alcoholmixture.

Fractions of 50 ml. volume were collected about every two hours, andfound to contain the. following:

Fractions l-8 (Nothing).

Fractions 9-17 (Compound S).

Fractions 18-19 (n- -d'ehydro-Compound S and l4u-hydroxy-Compound S);

Fractions 22-33 (l4a-hydroxy-Compound'S) The'fractions were evaporatedto dryness and the' crystals recovered; The n -dehydro-Compound S wastwice recrystallized from ethyl acetate, and the l4a-hydroxy-Compound Swas twice recrystallized from. ethyl alcohol. I i

n -dehydro-Compound S',. which may also be called .9 l7a,2l-dihydroxy-A-pregnadien-3,20-dione, has the following physical constants: M. P.235236 C., optical rotation am w 71.9

ultraviolet absorption maximum at 245 mn.

l4a-hydroxy-Compound S, which may also be called 14a,17a,2l-trihydroxy-A-pregnene-3,20-dione, is a white crystalline compound and has thefollowing physical constants: M.P. 226228 C., and optical rotationAnalysis.C, 69.64; H, 8.30. Calculated for C H O C, 69.57; H, 8.35.

EXAMPLE III An experiment was run exactly as outlined in the exampleabove except that 0.25 gram of desoxycorticosterone acetate Was used asthe starting material. After incubation for four days at 27 C. underotherwise the same conditions the product was recovered and analyzed bymeans of paper chromatograms. The following table indicates the results:

Rf Values-System Steroid Compounds Desoxycorticosterone acetateDesoxyccrticosterone 0. 97 A -dehydrodesoxycorticosteron 0.72 Amonohydroxy nuclear substitution prodnot of desoxycorfim terone 0. 44

Again in this example the products are formed by the nuclear oxidationof the steroid starting material, one of them in each example beingformed by oxygenation, and one in each example being formed bydehydrogenation at the 1,2 positions. The exact position of the hydroxylgroup in the monohydroxy nuclear substitution product here obtained isnot yet certainly known, except that it is in a different position fromthat of the nuclear hydroxyl group in corticosterone, and it is believedto be the 14 position.

EXAMPLE IV EXAMPLE V Submerged, aerated fermentation using progesteroneas the starting material was run with M. smegmatis ATCC. 278 in 4 literfermenters for 96 hours. The products obtained were A-dehydroprogesterone, and a product with a saturated A ring, along withsome unreacted progesterone. The mixture was separated by countercurrentdistribution. The physical constants for the A dehydroprogesteroneobtained here check with those reported in the literature.

EXAMPLE VI A culture of Mycobacterium smegmatis ATCC. 12,051, grown onsolid agar medium was rinsed into a sterile nutrient solution having thefollowing composition:

Solid, standard nutrient broth grams/liter 8 Glycerol mls./1iter 20Tween 80 mls./liter 0.2 Asparagine grams/liter One liter of inoculatedmedium was placed ineach of two Fernbach shake flasks. After three days,one-half gram of hydrocortisone was added to each flask. The reactionwas continued for four days at the end of which time the contents of theflasks were combined and extracted 3 times, each time with an equalvolume of chloroform. The combined chloroform extracts were concentratedto a volume of approximately 150 ml. A small amount of activated carbon(Nuchar) was added and the mixture was gently heated and stirred for afew minutes. The Nuchar was removed by filtration and the filtrate wasput on a silica gel-ethanol chromatography column. The column wasdeveloped, using a mixture of 97% by volume ethylene chloride and 3%ethanol. 50 ml. fractions were collected. The first 37 contained nosteroids. In fractions 38 through 66, unreacted hydro cortisone wasrecovered. Fractions 67 through 82 contained hydrocortisone and deltal-dehydrohydrocortisone. Fractions 83 through 115 contained deltal-dehydrohydrocortisone. Fractions 116 through 269 contained no steroid.Fractions 270 through 332 contained A preg nen-l 113, l7u,20,B,21-tetrol-3 -one.

Combined fractions 83 through 115 were evaporated to dryness on a steambath. The residue was dissolved in ethyl acetate and treated with asmall amount of activated carbon. The carbon was removed by filtration,and the filtrate concentrated to a small volume and refrigerated.Crystals which formed after refrigeration were redissolved in theminimum amount of acetone. An equal volume of hexane was then added,followed by the addition of cyclohexane to incipient turbidity. Onaddition al refrigeration, white crystals formed. These have beenidentified as being delta l-dehydrohydrocortisone, which may also becalled prednisolone or A -pregnadien-llfl, 17a,2l-t1i01-3,20-di0ne. Thecrystals had a melting point of l96198 C., a specific rotation of [M+108.2 in ethanol and EXAMPLE VII The procedure of Example VI wasrepeated except that cortisone was used in place of hydrocortisone. Theproduct, A -dehydrocortisone, which may also be called prednisone or A-pregnadien-17a,21-di0l-3,11,20-trione, was isolated in a similarfashion.

EXAMPLE VIII To a four liter Pyrex glass vessel equipped for conductingsubmerged aerated fermentation was added two liters of Turfitts medium.The aqueous medium was sterilized and then inoculated with ml. of aculture of Mycobacterium smegmatis ATCC 12,051, grown in a shake flaskon nutrient broth. After three days, 0.25 gram of hydrocortisone wasadded. The mixture was agitated and aerated with sterile air. Four daysafter the addition of the steroid, the whole mixture was extracted withapproximately 2 liters of chloroform.

Chromatography proved the presence of A -dehydrohydrocortisone in theproduct.

EXAMPLE IX The procedure of Example VIII was repeated except thatcortisone was used in place of hydrocortisone. In an identical manner,the product A -dehydrocortisone was prepared.

9 EXAMPLE X 5 7,5 scribed in corresponding application Serial Number476,-

11 556,@filed-;on;December 20, 1954, by G. Mushull et al.,

was added; The mixture was agitated and aerated with sterile air-. Fourdays after the addition of the steroidflche wh'ole mixture was extractedwith approximately 2 liters of chloroform; The chloroform extract wastreated with--activated carbon, filtered and reduced to a smallvolume byevaporation, and: then placed on a silica gel ethanol chromatographycolumn. The column was eluted withmixtures of ethylene chloride and 95%ethanol, starting with mixtures containing about 2% by volume ethanol,and gradually increasing the percentage of ethanol. The compound A-pregnadien-11fl,14a,17u,21- tetrol-3, 'dione was recovered in thisfashion. The physical. constants of this novel compound are as follows:M.P. 227229 0., [0519 +1035 (dioxane),

Infrared 1% in: 14811 pellet) most prominent absorption peaks at 2.92,5.78, 6.03, and 9.65 microns. This compound possesses great activity as.an adrenocortical a nutrient agar slant to-a Fernbach flask containing1000 cc. of the following medium:

Nutrient broth (Difco) g 8.0 Glycerol cc 20.0 Tween 80 (Atlas PowderCo.) cc 0.2

Distilled H O to make 1000.0 cc.

After 2 days shaking at 28", 100 cc. of the resulting broth wasus'ed toinoculate each-of four ferme'ntors containing-'2000'cc.-of Medium" CC.

Medium co, g. mane, 1.0 Kano, 0.25 MgSO -7H O 0.25 NaCl 0.005 resource0.0001. CaCO 5.0-

Distilled H O to 1000 cc.

Another 8' mg. was obtained as a second crop. Comparison of thiscompound with corticosterone in the liver glycogen and thymus involutionassays for glucocorticoids showed that the new compound was much moreactive than corticosterone. The compound was identified as A-pregnadiene-llflgZl-diol-B,20-dione, the expected transformationproduct.

I EXAMPLE Mycbliacte rium smegmat is ATCC 361' wastransferred from,anutrient: agar slant. to seven-,Fernbach flasks eacl of: whichcontained: 1000: cc. of the-following medium:

- G1 Malt extract (Difco) 510 Dextrose hydrate 30.0 NaNO 2.0.- K-H PO1.0 KCL I 0.5 MgSO JI-I Q 0.5- FeSO -7H 0 0.5 pH 6.7. Tap H O to 1000cc.

After 5 days shaking at 28, 250 mg. of 14a-hydroxy- Compound S was addedto each flask. After an additional 3 days, the fermentations werestopped and the broth extracted with CHClg. The combined extracts werechromatographed on silica gel to yield 33 mg. of a white crystallineproduct, M.PL 217-218, [ml (fl i);

xfig zss', 315-, 468, 52mm The compound has been identified as A'-pregnadiene- 14a,17a,2'1=triol-3,20-dione.

EXAMPLE XIV A scriesot experiments were run using the proceduresdescribed in-th'e above examples, and using the following.

species of Mycobaoteria, all of which are available from.

public culture collections:

M. phlei ATCC 10,142 M. thamnopheos M. ranae ATCC M. lacticola M-.butyricum M. friea'manni ATCC 114 M. berolinense ATCC M. tuberculosisother used steroids included the following:

A androstadiendione' 1 15,17a-hydroxyprogesterorie A -dehydro-Compound SH Androstenedione 19-nortestosterone l7a-hydroxyprogesterone 1 6dehydropro'g'esterone l'l -ketoprogesterone l' lmfiu-epoxiddCompound S(This compound may be obtained by the method describedin copendingapplication Serial Number 459,848, filed October 1, 19541) 14h,-15u-epoxido-Compound F (This compound may be obtained" by the methoddescribed in oopendi'ng application. Serial Number 432,621, filed May26, 1954".)

In-eachcase the products were recovered from the reaction mixture byextraction and were subjected to evaluation by the paper chromatographymethod. In each case, it was found that dehydrogenation at the 1-positi'on had occurred; What is claimed is; 1.1. Auprocess fortheprep'arationofa 3 ket0-A -stcroid compound which. process comprisescontacting a- 3-keto- At-steroid' compound having' from 18" to- 2 1atoms inthe carbon.- skeletonwithihe oxidizing enzymes of an organismof.theigenus Mycobacterium;

2. A process for'the preparation of'a 3'-lreto-A '-steroid compoundwhich process comprises subjecting a 3-keto- A -steroid having from- 18-to- 21. atoms in the carbon skeleton to submerged, aerated fermentationwith a living culture ofan organism of the genus Mycobacterium;

3 A processas claimed in claim 1 wherein the organ ism is-ofthefisp'ecies-Mycobacterium phlei.

4. A processasclaimed inclaim 1 wherein theorganism is of the-speciesMycobacterium smegmaris.

5. A process-as claimed in claim 2 wherein the organism is of;thespecies M ycqbacterium phlei.

6. A process as claimed in claim 2 wherein the organism is of thespecies Mycobacterium smegmatis.

7. A process for the preparation of A -pregnadiem11,8,17oz,21-tri013,20-di011e which process comprises contacting A-pregnene-11,8,17a,2-trio1-3,20-dione with the oxidizing enzymes of anorganism of the genus Mycobacterium.

8. A process for the preparation of A -pregnadien-17oc,21-d.i01-3,11,20-t1i0ne which process comprises contacting A-pregnene-17a,21-diol-3,11,20-trione with the oxidizing enzymes of anorganism of the genus Mycobacterium.

9. A process for the preparation of A -pregnadien-17a,21-dio1-3,20-dione which process comprises contacting A-pregnene-17u,21-dio1-3,20-dione with the oxidizing enzymes of anorganism of the genus Mycobacterium.

10. A process for the preparation of A -pregnadien-115,14a,17a,21-tetrol-3,20-dione which process comprises contacting Apregnene 115,14u,17a,21t6t1'01-3,20-di0116 with the oxidizing enzymes ofan organism of the genus Mycobacterium.

11. A process for the preparation of A -pregnadien-14a,17oc,21t1i01-3,11,20-13110116 which process comprises contacting Apregnene 14a,170c,21 trio1-3,11,20-tri0ne with the oxidizing enzymes ofan organism of the genus Mycobacterium.

12. A process for the preparation of A -pregnadien-11/8,17a,21-trio1-3,20-dione which process comprises contactingM-pregnene-l1,8,17a,21-trio1-3,20-dione with the oxidizing enzymes ofMycobacterium phlei.

References Cited in the file of this patent UNITED STATES PATENTS Murrayet a1 July 8, 1952 OTHER REFERENCES

